Melanogenic inhibitor, and methods of producing and using the same

ABSTRACT

A purified, naturally derived melanogenic inhibitor protein capable of inhibiting melanogenesis in pigmentary cells has an amino acid sequence SEQ ID NO: 4. A method of producing melanogenic inhibitor protein comprises grafting mammalian skin onto a live host, permitting the mammalian skin to remain on the host for a predetermined period of time, removing the mammalian skin, and extracting the protein from the skin. Methods of controlling melanogenesis in pigmentary cells or selectively destroying melanoma cells comprise the steps of mixing an effective amount of melanogenic inhibitor protein, or an active segment, derivative, or analog thereof, with a suitable carrier, and applying this mixture to the pigmentary or melanoma cells.

TECHNICAL FIELD

This invention relates to a melanogenic inhibitor, and methods ofproducing and using the same. More specifically, this melanogenicinhibitor comprises a naturally derived protein, or effective segmentsthereof, which reduces tyrosinase activity, inhibits pigmentary cellproliferation, and is cytotoxic to melanoma cells.

BACKGROUND ART

Hyperpigmentary disorders affect many people worldwide, and are oftenthe cause of much embarrassment or worse. These disorders include suchthings as: freckles, solar lentigines (livers spots), the visibledarkening that often occurs in skin grafted onto bum victims, and, mostserious of all, melanoma. Additionally, many also desire to lightentheir basal skin tone merely for cosmetic reasons.

Various means have been utilized in the past for either reducingpigmentation in hyperpigmented areas or for lightening basal skin tone.While mixed results have been obtained with these treatments, most canresult in undesirable side-effects. Compounds that have been used forbleaching, or depigmenting, skin include: hydroquinone, derivatives ofhydroquinone, ammoniated mercury, ascorbic acid, mercaptoamines,4-isopropylcatechol, and peroxides. None, however, have proven to becompletely reliable and without side effects.

European Patent Application 389,950 discloses a melanocyte-stimulatinghormone (MSH) inhibitor that is reported to be useful for such things aspreventing or relieving the symptoms of chloasmata or freckles. Thisinhibitor contains an amino acid sequence represented by the followingformulas: -His-Ser-Arg-Trp-; Trp-Arg-Ser-His-; or -Leu-Ala-Cys-Ala-Arg-.The peptides represented by the first two of these sequences have anaffinity for the MSH receptor contained on the surface of melanocytes,and thereby antagonize MSH. Peptides represented by the third sequencehave an affinity for MSH itself, and thereby inhibit the effect of MSH.

Subsequent to their invention, Applicants have become aware of a recentpublication which discloses a protein that may be related to that whichthey have produced (Madsen, P. et al., I. Invest Dermatol 99:299-305(1992)), and in fact may be identical. This protein is reported to behighly up-regulated in psoriatic keratinocytes, however no relationshipto melanogenesis is mentioned.

A more serious skin hyperpigmentary disorder that effects millionsworldwide is melanoma. While it can sometimes be treated if detectedearly, melanoma is often fatal. Currently available treatments, such aschemotherapy, often cause serious side effects, and are never completelyeffective in all instances.

Heretofore, however, there has not been an available naturally derivedcomposition that can reliably inhibit melanogenesis and thereby reduceskin pigmentation, is selectively cytotoxic to melanoma cells, and hasno known side effects.

SUMMARY OF THE INVENTION

While not exclusive, the following describes some of the importantfeatures and objects of the present invention.

It is an object of the present invention to provide a naturally derivedmelanogenic inhibitor useful for reducing pigmentation in skin andselectively destroying melanoma cells.

It is also an object of the present invention to provide a naturallyderived melanogenic inhibitor comprising a naturally derived melanogenicinhibitor protein, or active segments thereof.

It is a further object of the present invention to provide a method forproducing a melanogenic inhibitor protein.

It is yet another object of the present invention to provide a methodfor controlling pigmentation in skin and/or hair utilizing a naturallyderived melanogenic inhibitor protein, or active segments thereof.

It is another object of the present invention to provide a method fordestroying melanoma cells, without affecting normal, nonpigmentarycells, utilizing a naturally derived melanogenic inhibitor protein, oractive segments thereof.

As used herein, "melanogenesis" means the formation of melanin by livingcells, and "inhibiting melanogenesis" includes inhibiting the ability ofindividual cells to produce melanin and/or decreasing the population ofcells capable of producing melanin.

In accordance with one aspect of the invention, there is provided aheretofore unknown naturally derived protein capable of inhibitingmelanogenesis in pigmentary cells and selectively destroying melanomacells without harming other cells. This melanogenic inhibitor (MI)protein has the following amino acid sequence which is shown inconjunction with the corresponding codon (the numbers below each aminoacid refer to the location of the amino acid in the MI sequence):##STR1## For the sake of clarity, the sequence for the entire MI proteinis shown below: ##STR2##

In accordance with another aspect of the invention, there is provided amethod for producing MI protein comprising the steps of: (a) graftingmammalian skin, preferably human skin, onto a live nude mouse; (b)permitting the mammalian skin to remain on the mouse for a predeterminedperiod of time; (c) removing the mammalian skin from the mouse; and (d)extracting a protein portion containing MI protein from the mammalianskin. It is preferred that the mammalian skin graft be permitted toremain on the mouse for at least 6 weeks. It is also preferred that theextraction step comprise extracting proteins from the mammalian skinwith physiological saline, fractionating the skin protein extract usingion exchange chromatography, separating the skin extract proteins in theappropriate fraction obtained by chromatography using 2-dimensionalSDS-PAGE, and isolating MI protein from the electrophoresis gel usingelectroelution.

In accordance with yet another aspect of the invention, there is alsoprovided a method of controlling melanogenesis in pigmentary cells,especially those found in skin and hair, comprising the steps of forminga blend of an effective amount of MI protein and a suitable carrier, andthen applying this blend to the pigmentary cells to be controlled. MIprotein, or active segments thereof, can be applied to skin, forexample, to treat certain hyperpigmentary disorders or for merelycosmetic reasons. MI protein can also be applied to depigment or lightenhair for cosmetic reasons, such as for making unwanted body hair (e.g.,facial, underarm, chest, legs, arms, back) being less visible.Application of such compositions would be preferable to theinconveniences of shaving, depilation or electrolysis. less visible. Itis preferred that these blends be topically applied to the pigmentarycells, and that active segments of MI protein be employed. The entire MIprotein or active segments can, alternatively, be injected, along with asuitable carrier, in order to apply the composition to the pigmentarycells. The suitable carrier possibilities are endless, and their choicedepends upon the ultimate goal of the application. Thus, MI protein, ormore preferably an active segment of MI protein, could be combined witha skin repair agent in order to lighten aging spots while also improvingthe appearance of skin wrinkles.

In accordance with yet another aspect of the present invention, there isprovided a method of selectively destroying melanoma cells by mixing aneffective mount of MI protein, or an active segment thereof, with asuitable carrier, and applying this composition to melanoma cells isprovided. Once again numerous types of carriers can be employed, andtheir choice will depend upon the method of application of thecomposition. It is preferred that the composition be applied topically,and thus an active segment of MI protein should be utilized.

DETAILED DESCRIPTION OF THE INVENTION

A. MI Protein Developmental Work

Hyperpigmentation of skin grafted onto burn patients has been documentedpreviously, and was thought to be the result of a post-inflammatorymechanism. Increases in skin pigmentation have also been observed inconnection with sunburns and other skin inflammatory processes, andthese increases have been attributed to both an increase in the numberof functional melanocytes ("DOPA-positive" melanocytes) and an increasein the actual melanin synthesis by the melanocytes.

In order to examine pigmentary responses in skin grafts, small humanskin samples were grafted onto congenic athymic ("nude" or nu/nu) mice.Nude mice have proven to be an excellent host for studying the in vivobehavior of both normal and pathological xenograft tissues since theywill not reject grafts of distinct phylogenetical origin, and they havebeen used extensively in the study of human skin. Human skin graftedonto nude mice maintains its structural and immunological identity, andalso some of its major functional properties.

In this discussion and all of the examples that follow, viable donorskin was obtained from caucasian adult cadavers. The skin was removedusing a dermatome at a thickness of 12-14/1000th of an inch, and storedin F-12 tissue culture media (available from Gibco Laboratories, ofGrand Island, N.Y.) at 4° C. The nude mice were first anesthetized usingNembutol, and graft sites were prepared by removing the skin from theappropriate area of the torso. Split-thickness (12-14/1000th of an inch)donor skin was then positioned in the appropriate location on the miceand sutured in place. Grafts were attached to the mice within 48 hoursafter the death of the donor. When necessary, xenografts were removedfrom the mice using sterile surgical techniques, after the mice weresacrificed with an overdose of Nembutol.

As expected, the xenografts exhibited the same hyperpigmentation thathas been reported in bum patients. Various histological tests andexaminations were performed on the xenografts prior to the graftingitself, as well as at various times post-grating, in order to study thisobserved hyperpigmentation. Visible darkening of the xenografts occurredas early as two weeks post-grafting, and, although there were observabledifferences, all of the xenografts exhibited hyperpigmentation. Theobserved hyperpigmentation, however, was restricted to the xenograft,and did not extend into the host skin.

Microscopic analysis of xenograft samples revealed a significantincrease in the number of DOPA positive melanocytes in the xenografts ascompared to that noted prior to grafting. A DOPA positive melanocyte isone in which functional tyrosinase is present, and is thus producingpigment. While there was some variability in the extent of the increase,there was at least a three-fold increase in the number of DOPA positivemelanocytes, and all of the samples exhibited a peak at around six weekspost-grafting. From that point on, the number of DOPA positivemelanocytes steadily declined, however it remained slightly elevated insome xenografts as late as 30 weeks post-grating.

The size of the melanocytes cell bodies in the xenografts was alsomeasured as an indicator of melanogenic activity. In all cases, the sizeof the melanocytes more than doubled over that seen in the pre-graftingsamples, and once again exhibited a peak followed by a gradual decline.In addition, the dendricity of the melanocytes also markedly increasedin the xenografts. An increase in dendricity will generally result in anincrease in skin pigmentation, as the dendrites are responsible fortransferring melanin to the epidermis. While the degree ofhyperpigmentation did not always correlate with the number of DOPApositive melanocytes, it did tend to correlate with the dendricity ofthe melanocytes. This would tend to indicate that the observedhyperpigmentation is more a result of increased melanogenic activitythan an increase in the actual number of melanocytes.

SDS-PAGE analysis of both pre- and post-grafting human skin extract wasperformed in order to determine if there were any differences in bothlow and high molecular weight protein profiles. While there were noobvious differences in protein patterns between samples taken at varioustimes post-grafting, a protein band with an apparent molecular weight ofapproximately 13 to 14 kDa was exclusively expressed in thepost-grafting xenograft samples and was not found in the pre-graftedskin. Interestingly, a protein of similar molecular weight was alsofound in nude mouse skin extract. The 13 to 14 kDa protein band appearedas early as 2 weeks post-grafting, and continued to be expressed morethan 14 weeks post-grafting.

In the examples that follow, various protein fractions and purifiedproteins were applied to several different types of cells in order todetermine the effect on pigmentary function, if any, of proteinsextracted from both pre- and post-grafting skin. Pre-grafting skinsimply refers to normal human skin, while post-grafting skin refers tolive xenografts supported by nude mice. The culmination of this testinghas been the isolation of a unique Melanogenic Inhibitor (MI) proteinobtained from the 13 to 14 kDa band referred to above. While theapplicants are not certain as to MI protein affects melanogenesis andselectively destroys melanoma cells, there is reason to believe thatthese effects are mediated through MSH and/or its receptor.

EXAMPLE 1

The first cell type employed was Cloudman S91 melanoma, a moderatelymelanotic murine cell that has been used extensively to studymelanogenesis and pigmentary cell growth. The melanoma cells weremaintained in monolayer cultures in Ham's F-10 medium (available fromGibco Laboratories, of Grand Island, N.Y.) supplemented with 10%heat-activated mouse serum, 2.5% heat activated fetal calf serum, 100units/ml penicillin, and 100 μg/ml streptomycin, at 37° C. in a humifiedincubator containing 5% CO₂. Cells were subcultured weekly, andmaintained in the culture for only 10 passages in order to avoidphenotypic drift. Monolayer stock cultures were regenerated from cellsthat were cryopreserved in liquid nitrogen (as described by ZalfaAbdel-Malek et al., Cancer Research 47:3141-6 (1987)).

Protein extracts from both pre- and post-grafting (12 to 15 weeks aftergrafting) skin samples were prepared by first removing the underlyingfat and then cutting the skin sample into small pieces. A 20-30%homogenate in phosphate buffer saline (PBS) containing 1 mM PMSF wasprepared by homogenizing the skin using a polytron. After centrifugationat 2000 x g for 20 minutes, the crude extract was removed and furthercentrifuged at 100,000 x g for one hour to obtain a clear solublesupernatant fraction. The supernatant was filter sterilized, and theprotein content determined by Bio-Rad's method.

The Cloudman S91 melanoma cells were then harvested by replacing theculture medium with Tyrodes solution containing EDTA. Cells were seededat a density of about 0.2×10⁶ cells/25 cm² in each flask. Triplicateflasks were used for each experimental group, and control flasks werealso utilized. After 24 hours, this medium was replaced with freshmedium containing various concentrations of pre- or post-graftedsupernatant prepared as above, and the solutions were incubated for 24hours. Finally, fresh medium containing ³ H-tyrosine (1 μCi/ml) andsupernatant (in the same concentration as previously used) was added,and the solutions incubated for another 24 hours. The tyrosinehydroxylase activity of tyrosinase was then measured in situ accordingto a modified Pomerantz charcoal absorption method (as described byFuller, B. B. & Viskochil, B. H., Life Sci. 24:2405-2416 (1979)). Thisassay measures tyrosinase activity by measuring the amount of ³ H₂ Oreleased after ³ H-tyrosine is converted to L-DOPA by tyrosinase, and isa significant indicator of melanogenic activity since a reduction intyrosinase activity will result in a corresponding reduction inmelanogenesis (i.e. reduced skin pigmentation).

Melanoma cells treated with 25 μg/ml of post-grafting protein extractexhibited an increase in tyrosinase activity of approximately 20%, ascompared to the control. Concentrations less than 25 μg/ml did notincrease the tyrosinase activity any further. At higher concentrationsof post-grafting protein extract, however, the tyrosinase activity inthe melanoma cells was reduced in a dose-dependent fashion. Thereduction in tyrosinase activity was approximately 20% at a 50 μg/mlconcentration, 30% at 75 μg/ml, and 50% at 100 μg/ml. In contrast, themelanoma cell cultures treated with pre-grafted skin protein extract didnot exhibit any statistically significant change in tyrosinase activity.Thus, it is apparent that the production of a potent inhibitor ofmelanogenesis is induced when human skin is grafted onto a live host,and this inhibitor is present in the protein extract from thepost-grafting skin.

EXAMPLE 2

The effect of the protein extracts of Example 1 on normal humanmelanocytes was also examined. Normal melanocytes were obtained by firstremoving the subcutaneous tissue from the ventral surface of neonatalforeskin. The tissue was then incubated in 0.25% trypsin at 4° C.overnight. Next, the epidermis was manually split from the dermis, andboth were vigorously vortexed in growth medium. The supernatant was thenremoved and transferred onto a 25 cm² tissue flask (procedure furtherdescribed by Zalfa Abdel-Malek et al., Journal of Cellular Physiology,150:416-25 (1992)). The growth medium employed in both the extractionreferred to above and in the maintenance of the cells comprised: Ham'sF-10 medium, 10⁻⁴ M IBMX, 5% heat-inactivated fetal calf serum, 5%newborn calf serum, 5 μg/ml insulin, 2 μg/ml α-tocopherol, 2 μg/mltransferrin, 5 ng/ml TPA, 20 ng/ml cholera toxin, 10,000 units/mlpenicillin, and 10,000 μg/ml streptomycin. Once again the cells weremaintained at 37° C. in a humified incubator containing 5% CO₂.Melanocytes were then seeded into 6 cluster wells (9.6 cm² surface area)at a density of 0.15×10⁶ cells/well. The remaining steps of Example 1,including the extraction of pre- and post-grafting (12 to 15 weeks) skinprotein extracts, were the same.

Tyrosinase activity in the normal melanocyte cells was measured forvarious concentrations of protein extract treatments (25-100 μg/ml).Once again the same low-concentration increase in tyrosinase activityand subsequent dose-dependent decrease in tyrosinase activity at higherconcentrations of post-grafting protein extract was observed. The changein tyrosinase activity observed for each concentration was approximatelythe same as that recorded for the melanoma cells in Example 1, with a50% reduction in tyrosinase activity observed for cells incubated in 100μg/ml post-grafting protein extract. The cells treated with pre-graftedskin protein extract did not show any statistically significant changein tyrosinase activity. Thus, the melanogenic inhibitor present in thepost-grafting skin protein extract has identical inhibiting effects inboth normal melanocytes and melanoma cells.

EXAMPLE 3

In order to examine the various proteins contained in the pre- andpost-grafted skin protein extracts, one-dimensional 0.1% SDS-PAGEanalysis was performed. The discontinuous buffer system described byLaemmli (reported in Nature 227: 680-5 (1970)) was employed using a 15%acrylamide gel. Cells were stained with Coomassie brilliant blue R-250in order to visualize the protein bands. Both crude extract (2000 x g)and supernatant (100,000 x g) from pre- and post-grafting skin (preparedin the manner described in Example 1) were subjected to analysis.Additionally, the pellet remaining after the 100,000 x g centrifugationwas extracted with triton X-100 to solubilize any particulate fraction,and likewise subjected to SDS-PAGE.

No obvious differences were observed between the protein profiles orpre-grafted crude extract and supernatant. Likewise, there were also noobvious differences between post-grafting crude extract and supernatant.There were, however, significant differences between the proteinprofiles of pre- and post-grafting samples. Most significantly was theappearance of a 13 to 14 kDa doublet exclusively present in thepost-grafting skin samples. Furthermore, the fraction extracted from thepellet with triton X-100 did not contain this protein band, anindication that this band contains soluble protein(s). The dermis andepidermis of post-grafting skin were also split from one another, andthe steps above repeated. The 13-14 kDa protein band was present in bothsamples, however it was expressed to a greater extent in the epidermis.

The above analysis was also performed utilizing post-grafting skin thathad remained on its host for varying lengths of time. The 13-14 kDaprotein band appeared as early as 2 weeks post-grafting, and continuedto be expressed more than 14 weeks post-grafting.. No significantdifferences were noted in protein patterns even between sample from 2and 14 weeks post-grafting.

EXAMPLE 4

In order to determine if the 13-14 protein band comprises multipleproteins, two-dimensional polyacrylamide gel electrophoresis wasperformed on both pre- and post-grafting skin supernatant. Thesupernatant extract from post-grafting skin (obtained in the mannerdescribed in Example 1) was first, however, subjected to ion-exchangechromatography using a DEAE-cellulose column (15cm×1.5 cm). Thesupernatant (15 ml) was loaded onto the column, and the column waswashed with phosphate buffer to elute the unbound proteins. Fractionscontaining 5ml of solution were taken every 5 minutes, and theabsorbance at 280 nm was measured for each collected fraction. Thecolumn was washed with phosphate buffer until the absorbance of theeluting fractions was near negligible. Thereafter, the bound proteinswere eluted from the column by a salt gradient (NaCl in phosphatebuffer) of 0 to 1.0M. Spectroscopic analysis of the fractions indicatedthe presence of two distinct absorbance peaks (at 280 nm).

Next, the fractions associated with each of the two peaks were pooledand dialyzed extensively against double distilled water containing 0.02%sodium azide for at least 36 hours. The dialyzed peaks were thenlyophilized overnight to obtain dried powder. The powders were thendissolved in distilled water and protein contents determined. These twopeak fractions were then each subjected to two-dimensional SDS-PAGEanalysis.

In order to perform two-dimensional SDS-PAGE analysis, aliquots of thetwo peak fractions containing 700-800 μg of protein were lyophilized.The two samples were then solubilized in 9M urea containing 2% CHAPSdetergent (available from Sigma Chemical Co.). and 2% each of ampholine(pH 3-10) and β-merceptoethanol, and incubated for 2 hours at roomtemperature. After centrifugation at 14,000 rpm for 15 minutes, thesamples were loaded onto isoelectric focusing gels. Isoelectric focusingwas carried out using ampholine pH 3-0 overnight at 700 volts (asdescribed by Farrell, J. Biol. Chem. 250: 4007-21, (1975)). The gelswere then equilibrated in SDS-equilibrating buffer for at least 15minutes before loading into the slab gel for second-dimensional SDS-PAGEusing 15% gel. The gels were stained with Coomassie blue to visualizethe various protein spots.

In the supernatant samples obtained from post-grafting skin, the 13-14kDa protein band of the first absorbance peak fraction was resolved intofour distinct protein spots having pI (isoelectric point) values in therange of 6.5 to 7.5. The second absorbance peak solution, however, didnot exhibit any proteins in this molecular weight range. Likewise, whenthe preceding steps were repeated for pre-grafted skin, these proteinswere not expressed. Thus, it is apparent that the 13-14 kDa protein bandfound in post-grafting skin actually comprises four distinct proteins.

EXAMPLE 5

In order to isolate and test the four proteins contained in the 13-14kDa protein band, electroelution was employed. Unstained gels obtainedby the one-dimensional SDS-PAGE procedures of Example 3 usingpost-grafting skin supernatant were aligned side by side with stainedgels (as a marker), and the 13-14 kDa protein band was cut from theunstained gels. The gels were then cut into small pieces and transferredinto the electroelution chamber of a Schleicher and Schuellelectroelution apparatus (available from S.S. Inc., Keene, N.H.) Abuffer system comprising 20 mM Tris, 150 mM glycine, and 0.05% SDS wasadded, and electroelution was carried out overnight at 150 volts. Theelectroeluted fractions were then collected, concentrated and dialyzedusing a Centricon-3 concentrator (available from Amicon Corp., divisionof W.R. Grace & Co., Beverly, Mass.) to a volume of about 0.5 ml.Finally, this concentrated 13-14 kDa protein mixture was filtersterilized and its protein contents determined.

The procedures of Example 1 were followed in order to examine the effecton tyrosinase activity in melanoma cells of the electroeluted proteins.The 13-14 kDa protein mixture obtained from post-grafting skinsupernatant was added to the cell cultures in concentrations rangingfrom 0.25 to 1.0 μg/ml. In order to ensure that any observed effectswere indeed due to the protein mixture present in the 13-14 kDa bandfrom post-grafting skin, the entire procedure was also performed forsupernatant from pre-grafted skin.

The 13-14 kDa protein mixture obtained from post-grafting skin causedrather significant dose-dependent reductions in tyrosinase activity inthe melanoma cells. In fact, this reduction was almost 90% at a proteinconcentration of 1.0 μg/ml. In contrast, the tests using correspondingamounts of solutions obtained from pre-grafted skin supernatant resultedin only slight, insignificant reductions in tyrosinase activity.

When these same tests were repeated using normal human melanocytes(obtained by the methods of Example 2), the 13-14 kDa protein mixturelikewise caused a reduction in tyrosinase activity. At a concentrationof 0.25 μg/ml, a reduction in tyrosinase activity of approximately 37%was observed; at 0.5 μg/ml, the observed reduction was approximately53%. Very little reduction of tyrosinase activity was observed whenextract from pre-grafted skin was employed. These results indicate thatthe melanogenic inhibitor present in post-grafting skin extract iscontained in the 13-14 kDa electroeluted protein mixture.

EXAMPLE 6

The effect of pre- and post-grafting skin extracts on cell proliferationwas assessed by measuring the rate of ³ H-thymidine incorporation, whichis a measurement of the rate of DNA synthesis within the cell.Obviously, a reduction in melanocyte proliferation will cause acorresponding reduction in observed skin pigmentation, since themelanocytes are directly responsible for pigment production. Cloudmanmelanoma cells were seeded into a 96-well flat-bottom culture plate in avolume of 200 μl/well. Cells in F-10 growth medium were permitted toattach, and on the following day half of the volume of medium wasreplaced with 100 μl fresh medium and a selected amount of skin proteinextract. Each experimental group consisted of 6 wells. After 24 hours oftreatment, half the medium was again I0 replaced with fresh medium andskin protein extract. During this latter treatment period, the cellswere pulsed labeled with ³ H-thymidine (1 μCi/well).

After 24 hours incubation, the cells were harvested onto glass fiberfilters using a semi-automated PHD cell harvester (available fromCambridge Technology). The filters were air dried overnight, transferredindividually to scintillation vials, and treated with 450 μl Protosoltissue and gel solubilizer. The vials were then warmed in an oven at 66°C. for 75 minutes, then allowed to cool. Next, 100 μl of glacial aceticacid and 9.5 ml EconoFluor-2 (a non-aqueous scintillant) were added toeach vile. The viles were allowed to cool in a refrigeratedscintillation counter for 24 hours, and counts were then taken for oneminute.

In the samples employing the 13-14 kDa protein mixture (electroeluted asin Example 5), a dose-dependent inhibition of melanoma cellproliferation (as compared to control samples) was observed. Thisinhibition was more than 30% at a protein mixture concentration of 0.25μg/ml, and was nearly 50% at a concentration of 0.50 μg/ml. In contrast,melanoma cells treated with extract from pre-grafted skin exhibited aslight, insignificant reduction in cell proliferation. Normal humanmelanocyte cell proliferation was also inhibited by the 13-14 kDaprotein mixture to the same extent when these tests were repeated withnormal melanocytes. Likewise, the pre-grafted skin extract did notinhibit cell proliferation in normal melanocytes.

Melanoma cells were also observed after the incubation period under aphase contrast microscope in order to study their morphology. In theuntreated cells (control) and those treated with extract frompre-grafted skin, the cells appeared to be normal and healthy, withprominent dendrites extending between cells and forming thecharacteristic network. In contrast, the cells treated with the 13-14kDa protein mixture were not healthy, and numerous non-attached,floating dead cells were present. In fact, only about 40% of the cellsremained viable, and cell density was greatly reduced. Additionally,very few dendrites extended from the remaining cells, and virtually allwere round in shape. This is a clear indication that, not only does themelanogenic inhibitor inhibit pigmentary cell proliferation, it is alsocytotoxic to melanoma cells.

EXAMPLE 7

In order to determine if the 13-14 kDa protein mixture isolated frompost-grafting skin was merely a general metabolic inhibitor, ³H-thymidine cell proliferation testing was repeated using normal humanfibroblasts. Normal human diploid neonatal foreskin fibroblasts werepurchased from Clonetics Corporation, and, upon arrival, they were fedDulbeeco's Modified Eagle Medium (DMEM) supplemented with 10% fetalbovine serum in T-25 flasks. After three days of incubation at 37° C.,the fibroblasts were trypsinized and seeded into 48-well microtiterplates at 3,000 cells per square centimeter. The plates were thenincubated at 37° C. for three days. Next, the media was changed to 0.2%serum in DMEM and the cells remained in this environment for 4 to 5 dayswith no other additions.

Two types of assays were performed: agonist and antagonist. In theagonist assay, various concentrations of the post-grafting skin proteinextract were added to randomly selected wells. Fibroblast Growth Factor(FGF) was used as a positive control. Tritiated thymidine (to studyproliferation) or tritiated proline (to study collagen production) wasalso added. The cells were incubated in this media for 2 days(proliferation) and 4 days (collagen production), respectively. Themedia was then removed and the cells washed and then lysed with SDS. Oneml of this lysate was added to 9 ml of scintillation cocktail andcounted. For the antagonist assays the same procedure was followedexcept that FGF was also added to each well that contained the proteinmixture to determine if the active component of the mixture competedwith FGF and displayed a resultant decrease in tritiated thymidine ortritiated proline incorporation. The protein extract exhibited no effecton proliferation and collagen production in either the agonist orantagonist assays, regardless of concentration. Therefore, it isapparent that the effects of the 13-14 kDa protein mixture are specificfor pigmentary cells, and the melanogenic inhibitor present in thismixture is not merely a general metabolic inhibitor.

EXAMPLE 8

Since the studies described in Example 4 indicated that themelanogenesis inhibiting 13-14 kDa protein mixture actually comprisesfour distinct proteins, tests were undertaken to determine which ofthese proteins was responsible for the observed effects. Post-gratingskin supernatant was fractionated using a DEAE-cellulose ion exchangecolumn as in Example 4. The fractions corresponding to the first peak(as discussed in Example 4) were collected and pooled, and the mixturewas then dialyzed and lyophilized. Two-dimensional SDS-PAGE, asdescribed in Example 4, was then performed in order to separate theprotein mixture into the four distinct proteins previously found. Thegels were then stained with Coomasie blue, and the four protein "spots"obtained were individually cut from the gels. Each of the four proteinswere then electroeluted from the gels (as described previously) andconcentrated. Protein contents of the resulting solutions were alsodetermined. In this manner, the four purified (isolated from each other)proteins were obtained.

Both tyrosinase activity and ³ H-thymidine incorporation in Cloudmanmelanoma cells were studied using the procedures outlined previously. Aprotein concentration of approximately 1.0 μg/ml of each of the fourpurified proteins was utilized in these tests, and, as controls, bothuntreated cell cultures and α-MSH (10⁻⁷ M) treated cultures wereincluded in the experiment. Tyrosinase activity was reduced byapproximately 40% in the cultures treated with purified proteincorresponding to the third protein "spot". Cultures treated with theother three proteins, on the other hand, did not exhibit any significantdecrease in tyrosinase activity. Similarly, cell proliferation wassignificantly inhibited (approximately 45%) in the presence of thepurified protein corresponding to the third "spot", while the otherproteins had no significant effect on cell proliferation.

Based upon these tests, it is apparent that the protein electroelutedfrom the third gel "spot" is responsible for the melanogenesisinhibition and cell proliferation inhibition previously observed. Theprevious two-dimensional SDS-PAGE analysis indicated that thisMelanogenic Inhibitor ("MI") protein corresponding to the third "spot"has a molecular weight of approximately 14,000, and an isoelectric pointof between about 7.2 and about 7.5.

EXAMPLE 9

To determine the amino acid sequence of the four proteins contained inthe 13-14 kDa protein mixture, two-dimensional SDS-PAGE analysis wasonce again performed on the fractionated post-grafting skin extract inthe manner described previously. Proteins were blotted onto ImmobilonPSQ (a PVDF membrane) and visualized after staining with Amido black.Each of the four "spots" was then excised from the blot and reacted withiodoacetamide before sequencing. Protein sequencing was performed on anApplied Biosystems 475A protein sequencer using pulsed-liquid chemistry(Speicher, D. W., (1989), Techniques in Protein Chemistry, (T. E. Hugh,Ed.), Academic Press, San Diego, Calif., pp. 24-35).

Partial N-terminal amino acid sequence analysis of the first "spot"indicated that this protein is identical to mouse transthyretin, orprealbumin. Transthyretin is a serum protein having a molecular weightof 55 kDa, however it is composed of four identical subunits having amolecular weight of approximately 14 kDa. The sequence analysis of thesecond "spot" indicates that it has a strong homology with a chain ofhemoglobin. The sequence of the fourth "spot" has little or no homologywith any known proteins.

Since N-terminal amino acid sequence analysis of the intact MI protein(third "spot") indicated that the N-terminus of the protein was blocked,it was decided to fragment the protein using a protease and thensequence the resulting peptides. MI protein from four Immobilon PSQblots was cut out, reduced, alkylated with iodoacetamide, and digestedwith endoproteinase Lys C (Stone, K. L., M. B. LoPresti, J. M. Crawfrod,R. DeAngelis and K. R. Williams, (1989), A Practical Guide to Proteinand Peptide Purification for Microsequencing, (P. T. Matsudaira, Ed.),Academic Press, San Diego, Calif., pp. 31047).

Sequencing of the second peak of MI protein obtained after proteolysisof MI protein was more successful, and the following amino acid sequencewas obtained: ##STR3## The sites indicated by an "Xaa" are those inwhich either an identification could not be confirmed or could not bemade at all. Tentative assignments of Cys to site number 5, and Asp tosite 19 were made, however. Interestingly, 9 of the 12 C-terminal aminoacids in this protein segment correspond to a region of mouse fatty acidbinding protein (mFABP), however no significant homology was found forthe N-terminal half of the segment.

Sequencing of the third peak obtained from MI protein was as follows:##STR4## Additionally, a tentative assignment of Ile to site number 15was made. In this case, 13 of the 18 amino acids in this segmentcorrespond to a region of mFABP, with only conservative changes at the12th, 15th, and 18th amino acids.

Based on this sequencing, it is apparent that MI protein is highlyrelated to mFABP, however it is definitely not identical to it. Mousefatty acid binding protein is a member of a family that includes fattyacid binding proteins, retinoic acid binding proteins, adipocytedifferentiation proteins, and myelin P2 proteins, and, based upon thetwo sequences identified, it is expected that MI protein is also amember of this family. Thus, since most of the proteins in this familycomprise about 131 amino acids, it is further expected that MI proteincomprises about 131 amino acids. The fact that MI protein has a closehomology to a known protein such as mFABP also is a strong indicationthat, as the applicants' studies have shown, it is a biologicallyrelevant molecule.

EXAMPLE 10

In order to determine the entire sequence of the MI protein, appropriateoligonucleotide probes for the MI protein were prepared. These oligoswere as follows:

5'- CAG CCC GCC CGC ACC -3'

5'- AAA AAA GAA AGA AAC AGT ATG -3'

These oligos were utilized in performing a polymerase chain reaction(PCR) on RNA obtained from human skin which had previously been graftedonto nude mice in the manner previously described. The PCR reactionresulted in the formation of a considerable amount of single-size DNAwhich codes for MI protein. The isolated DNA was then cloned into asequencing vector and its sequence determined by known methods. Thenucleic acid sequence was as follows, and the corresponding amino acidsequence of MI protein is also shown below the corresponding codons(numerals indicate the position of the amino acids in MI protein):##STR5## For the sake of clarity, the sequence for the entire MI proteinis shown below: ##STR6##

In order to verify that SEQ ID NO: 4 was indeed MI protein, a proteinwas recombinantly expressed from SEQ ID NO: 4 using well-known methods.The effect of the recombinantly expressed protein on melanocyteproliferation was then determined by examining the rate of ³ H-thymidineincorporation in a fashion similar to that of Example 6. A significantdose-dependent inhibition of melanocyte proliferation was observed,clearly indicating that the protein recombinantly expressed from nucleicacid sequence SEQ ID NO: 3 is indeed MI protein. Therefore, thestructure of MI protein is shown by SEQ ID NO: 4. As those skilled inthe an will recognize, SEQ ID NO:3 can also be employed in well-knownmethods to verify that any product prepared according to Applicants'synthesis method is indeed MI protein.

EXAMPLE 11

In order to examine the in vivo effects of MI protein on hyperpigmentedskin grafts, the entire 13-14 kDa protein mixture containing MI wasinjected into human skin that had previously been grafted onto nude micein the manner described previously. Seven mice were utilized in thesetests, and these mice had supported the human skin graft for a period of10 to 12 months prior to the testing. Various degrees ofhyperpigmentation were present in the xenografts treated.

The mice to be subjected to the testing were subdivided into anexperimental group consisting of five mice, aid a control groupconsisting of the remaining two mice. The mice in the control group werenot given any injections. In the experimental group, injections weremade subcutaneously into the left side of the human skin graft at weeklyintervals over a five week period. The dosage in each injectionconsisted of a 50 μl solution containing 2 μg of the protein mixturewith the remainder of the solution consisting of normal saline. As aninternal control, 50 of normal saline was injected into the right sideof the graft at the same intervals. One week after the first set of fiveinjections, the entire testing protocol was repeated. For the second setof five injections, however, the site of injection was changed. Duringthis period, the protein mixture was injected into the dorsal area ofthe graft and the normal saline (control) was injected into the ventralarea of the graft.

Biopsies were taken from the skin grafts before the series ofinjections, one week after the first set of five injections (but priorto the second set of injections), and after the final injection. Thesebiopsies were taken near the point of injection of both the proteinfraction and the normal saline. Biopsies were also taken from thecontrol group.

The number of DOPA positive melanocytes in each biopsy were counted, andthe overall results for the testing are shown in Table 1 below. Thenumber of DOPA positive melanocytes is a direct measure of tyrosinaseactivity within the melanocytes themselves, and the results shown inTable 1 indicate that the protein mixture containing MI protein reducestyrosinase activity. The reduction in number of DOPA positivemelanocytes ranged from 9.0% up to 32.3%. No significant reductionoccurred in the biopsies taken from either the control group or from thelocations where normal saline was injected into the experimental group.

Visual examination of the mice indicated a marked reduction inhyperpigmentation, particularly at the site of injection, after just twoinjections of the protein mixture. No such reduction was seen in eitherthe control group, or at the sites of normal saline injection in theexperimental group.

                  TABLE 1                                                         ______________________________________                                                   % Reduction in Number of DOPA                                                 Positive Melanocytes                                                            Protein Mixture                                                                          Saline Injection                                      Mouse        Injection Sites                                                                          Sites                                                 ______________________________________                                        A            32.3       2.5                                                   B            29.0       1.3                                                   C            24.6       0                                                     D            9.0        0                                                     E            10.1       2.0                                                   F - Control  0          0                                                     G - Control  0          0                                                     ______________________________________                                    

Sections of several biopsies were also stained with hematoxylin andeosin (H&E) and viewed microscopically in order to examine anymorphological changes resulting from the injections of the proteinmixture. The experimental and control groups were indistinguishable fromone another, and neither group exhibited any evidence of injury ortoxicity to either the epidermis or dermis. Thus, MI protein is capableof reducing hyperpigmentation without causing undesired side effects.

EXAMPLE 12

In order to examine the effects of MI protein in preventing or delayingthe hyperpigmentation which normally occurs in skin grafts immediatelyafter grafting, the protein mixture utilized in Example 11 was alsoinjected into nude mice which had only supported a human skin graft for2 weeks. The protocol of Example 11 was followed, except that only thefirst set of five injections were performed. The protein mixture wasinjected into the dorsal area of the skin graft, and normal saline wasinjected into the ventral area. Initial counts of DOPA positivemelanocytes were performed just prior to the first injections.

Visual examination of the mice revealed some reduction in pigmentationat the site of protein mixture injection. More importantly, as shown inTable 2, the number of DOPA positive melanocytes was once againsignificantly reduced in the biopsies taken from the areas at the siteof protein mixture injection. In contrast, as expected, the number ofDOPA positive melanocytes increased in the biopsies taken from thecontrol group and from the areas at the site of saline injection in theexperimental group. This increase ranged from 190% to 300%, while thereduction caused by the protein mixture containing MI ranged from 8.3%to 31.6%. There was also no evidence of toxicity in the mice injectedwith the protein mixture. This data confirms that MI protein can bothprevent or reverse hyperpigmentation, most likely by altering tyrosinaseactivity, without affecting cell viability.

                  TABLE 2                                                         ______________________________________                                                   % Change in Number of DOPA                                                    Positive Melanocytes                                                            Protein Mixture                                                                          Saline Injection                                      Mouse        Iniection Sites                                                                          Sites                                                 ______________________________________                                        H            -31.6      +230                                                  I            -17.5      +300                                                  J            -11.2      +210                                                  K            -27.5      +190                                                  L             -8.3      +200                                                  M - Control  >200       +240                                                  N - Control  >200       +228                                                  ______________________________________                                    

EXAMPLE 13

The in vivo effects of MI protein on normal pigmented mice (C57BL/6) wasalso examined. This particular type of mouse has proven useful forstudying pigmentation and hair growth, since the truncal skinpigmentation in this mouse originates in the melanocytes of the hairfollicles and not the epidermis. Once these hair follicles are plucked,the underlying skin becomes less pigmented due to the reduction inmelanocytes. Although the hair follicles resume growth immediately afterplucking, repigmentation of the skin does not occur until five or sixdays later. Thus, this mouse is a good model for examining the effect ofMI protein in delaying repigmentation.

Five C57BL/6 mice were employed, and the hair follicles were pluckedfrom small areas on the fight and left dorsal side of each. The proteinmixture utilized in Example 11 (50 μl containing 2 μg protein mixture)was injected subcutaneously into the left dorsal area of each mousewhere the hair follicles had been removed. As a control, anelectroeluted 13 to 14 kDa protein fraction obtained from normal humanskin (prior to any grafting) was injected into the right dorsal area ofeach mouse. This protein fraction from normal human skin was obtained inthe same manner as that described previously. The injections were givenonce a day for five days. Photographs were taken each day to monitorskin color changes, and biopsies were taken from both the right and leftdorsal portions of each animal.

The left dorsal area into which the protein mixture containing MIprotein had been injected showed a reduction in skin color after only 2injections. No significant reduction in skin color was observed in theright dorsal area. Additionally, the appearance of new hair follicleswas also delayed in the left dorsal areas as compared to the right(control).

H&E staining patterns appeared normal for the biopsies from both theright and left dorsal areas, and no morphological differences betweenthe two were observed. There was also no evidence of toxicity, howeverthe biopsies from the left dorsal area into which the protein mixturecontaining MI protein had been injected showed a reduced staining ofmelanin in the hair follicles (as shown by DOPA staining).

B. Pharmaceutical and Cosmetic Compositions and Methods

As used herein, "topical application" means directly laying on orspreading on outer skin, "cutaneous injection" means introduction of asubstance beneath or within the skin by a hypodermic needle, and"comprising" means that other steps and other ingredients which do notaffect the end result can be added. This latter term thus encompassesthe terms "consisting of" and "consisting essentially of".

The present invention further relates to a composition comprising a) asubstantially pure protein for inhibiting melanogenesis in pigmentarycells, or an active melanogenesis inhibiting segment, derivative oranalog thereof, said protein having a molecular weight of about 14,000,an isoelectric point of between about 7.2 and about 7.5, and amino acidsequence SEQ ID NO.: 4; and b) a cosmetically- orpharmaceutically-acceptable carrier. In one embodiment of the invention,the carrier is an injectable carrier. In another embodiment of theinvention, the carrier is a topical carrier.

The compositions of the present invention comprise a solid, semi-solidor liquid cosmetically and/or physiologically acceptable carrier toenable the MI protein, or active segment, derivative, or analog thereof,to be delivered to the desired target at an appropriate concentration.The carrier can itself be inert or it can possess physiological orpharmaceutical benefits of its own. The nature of the carrier will bedictated by the method chosen for administration of the composition. Asafe and effective mount of carrier is preferably from about 50% toabout 99.9999%, more preferably from about 90% to about 99.9% of thecomposition. Variations in formulation of these carriers will result ina wide variety of products which fall within the scope of the presentinvention. The method of administration of the MI protein, or activesegment thereof, composition may range from internal methods such asinjection to external topical methods.

A preferred method of administration of the MI protein, or activesegment thereof, is by cutaneous injection. The carrier for facilitationof such administration would preferably comprise water or a salinesolution, preferably an isotonic saline solution.

A more preferred method of administration of the MI protein or activesegment thereof is by topical application. The topical pharmaceuticalcompositions of the present invention may be made into a wide variety ofproduct types. These include, but are not limited to lotions, creams,beach oils, gels, sticks, sprays, ointments, pastes, mousses andcosmetics. These product types may comprise several types of carriersystems including, but not limited to solutions, emulsions, gels andsolids.

Topical pharmaceutical compositions of the present invention furthercomprise from about 2% to about 50% of a topicalpharmaceutically-acceptable emollient. As used herein, "emollients"refer to materials used for the prevention or relief of dryness, as wellas for .the protection of the skin. A wide variety of suitableemollients are known and may be used herein. Sagarin, Cosmetics, Scienceand Technology, 2nd Edition, Vol. 1, pp. 32-43 (1972), incorporatedherein by reference, contains numerous examples of suitable materials.Particularly useful emollients which provide skin conditioning areglycerol, hexanetriol, butanetriol, lactic acid and its salts, urea,pyrrolidone carboxylic acid and its salts, amino acids, guanidine,diglycerol and triglycerol.

The present invention further relates to a method for inhibitingmelanogenesis in mammalian skin and/or hair. Such a method comprisestreating the skin and/or hair with a safe and effective amount of the MIprotein, or active segment, derivative or analog thereof. The amount ofMI protein, or active segment thereof, and frequency of treatment willvary widely depending upon the level of pigmentation and/ormelanogenesis already in existence in the subject and the amount ofmelanogenesis inhibition desired.

A preferred method of treating the skin and/or hair is via cutaneousinjection of a safe and effective amount of the MI protein, or activesegment thereof, to inhibit melanogenesis in mammalian skin and/or hair.The carrier for injectable administration of the MI protein, or activesegment thereof, would preferably comprise water or a saline solution.The amount of MI protein, or active segment thereof, and the frequencyof cutaneous injection can vary widely, depending on personal needs. Asan example of treatment by cutaneous injection, it is suggested that acomposition suitable for cutaneous injection comprising the MI protein,or active segment thereof, be cutaneously injected from once per day toonce every six months, preferably from three times per week to once permonth, more preferably from once per week to twice per month. Thecomposition for cutaneous injection will contain from about 0.0001% toabout 10%, preferably from about 0.001% to about 5%, more preferablyfrom about 0.01% to about 1% of the MI protein. The period of injectionswould be over a period of from about one month to about ten years,preferably from about three months to about two years, more preferablyfrom about six months to about one year, thereby resulting in inhibitionof melanogenesis in mammalian skin and/or hair.

A more preferred method of treating the skin and/or hair is via topicalapplication of a safe and effective amount of the MI protein, or activesegment thereof, to inhibit melanogenesis in mammalian skin and/or hair.The amount of MI protein, or active segment thereof, and frequency oftopical application to the skin and/or hair can vary widely, dependingupon personal needs, but it is suggested as an example that topicalapplication range from about once per week to about 10 times daily,preferably from about twice per week to about 4 times daily, morepreferably from about 3 times a week to about twice daily, mostpreferably about once per day. The composition for topical applicationwill comprise from about 0.001% to about 20%, preferably from about0.01% to about 10%, more preferably from about 0.1% to about 5% of theMI protein, or active segment, derivative or analog thereof. The periodof topical application would preferably be over a period of from aboutone month to about ten years, more preferably from about three months toabout two years, more preferably still from about six months to aboutone year, thereby resulting in inhibition of melanogenesis in mammalianskin and/or hair.

The following examples further describe and demonstrate certainpreferred embodiments within the scope of the present invention. Theexamples are given solely for the purpose of illustration, and are notto be construed as limitations of the present invention since manyvariations thereof are possible without departing from its spirit andscope. These prospective example merely demonstrate how the melanogenicinhibitor of the present invention may be employed.

EXAMPLE 14

An oil-in-water emulsion is prepared by combining the followingcomponents utilizing conventional mixing techniques.

    ______________________________________                                                                Percent by Weight                                     Component               of Composition                                        ______________________________________                                        Deionized water         Quantum sufficit                                      Glycerin                3                                                     Methyl paraben          0.2                                                   MI protein              0.1                                                   Steareth 20 (Brij 78R)  1                                                     Glyceryl monostearate and PEG 100 (Arlacel 165R)                                                      0.5                                                   Carbopol 940 (B. F. Goodrich, Cleveland, OH)                                                          0.2                                                   99% triethanolamine     0.2                                                   Cetyl alcohol           1.0                                                   Stearyl alcohol         1.0                                                   Propyl paraben          0.1                                                   Diisiopropyldimerate    2.0                                                   C12-C15 alcohol benzoate                                                                              6.0                                                   Imidazolidinol urea     0.3                                                   ______________________________________                                    

This composition is useful for topical application to inhibitmelanogenesis in skin and/or hair. An amount of the compositionsufficient to deposit about 0.01 mg/cm² of the MI protein to the skinand/or hair follicles is used. The composition is applied once per dayfor the subject's lifetime.

EXAMPLE 15

A clear gel is prepared by combining the following components utilizingconventional mixing techniques.

    ______________________________________                                                               Percent by Weight                                      Component              of Composition                                         ______________________________________                                        Deionized water        Quantum sufficit                                       Carbopol 980 (B. F. Goodrich, Cleveland, OH)                                                         0.5                                                    Disodium EDTA          0.02                                                   SEQ ID NO: 1           0.5                                                    99% triethanolamine    0.5                                                    Propylene glycol       3.0                                                    Methyl paraben         0.2                                                    ______________________________________                                    

This composition is useful for topical application to inhibitmelanogenesis in skin and/or hair. An amount of the compositionsufficient to deposit about 0.01 mg/cm² of MI protein active segment tothe skin and/or hair follicles is used. The composition is applied threetimes per day over a six-month period.

EXAMPLE 16

An oil-in-water polymer emulsion is prepared by combining the followingcomponents utilizing conventional mixing techniques.

    ______________________________________                                                               Percent by Weight                                      Component              of Composition                                         ______________________________________                                        Deionized water        Quantum sufficit                                       Carbopol 954 (B. F. Goodrich, Cleveland, OH)                                                         0.2                                                    Pemulen TR-2 (B. F. Goodrich, Cleveland, OH)                                                         0.15                                                   Glycerin               3.0                                                    SEQ ID NO: 2           0.75                                                   99% triethanolamine    0.35                                                   Cetyl palmitate        2.0                                                    Stearoxy trimethyl silane and stearyl alcohol                                                        1.0                                                    Squalane               6.0                                                    Propyl paraben         0.1                                                    Methyl paraben         0.2                                                    Imidazolidinol urea    0.3                                                    ______________________________________                                    

This composition is useful for topical application to inhibitmelanogenesis. An amount of the composition sufficient to deposit 0. 1mg/cm² MI protein active segment to the skin and/or hair follicles isused. The composition is applied once per week over a one-year period.

EXAMPLE 17

An oil-in-water microemulsion is prepared by combining the followingcomponents utilizing conventional mixing techniques.

    ______________________________________                                                              Percent by Weight                                       Component             of Composition                                          ______________________________________                                        Deionized water       Quantum sufficit                                        MI protein            1.0                                                     PEG4 sorbitan monolaurate                                                                           22.5                                                    PEG5 sorbitan monolaurate                                                                           2.5                                                     Cetearyl octanoate    25.0                                                    DMDM hydantoin and 3-iodo-2-propynyl                                                                0.2                                                     butyl carbamate (glydant plus)                                                ______________________________________                                    

This composition is useful for topical application to inhibitmelanogenesis. An amount of the composition sufficient to deposit 0.4mg/cm² MI protein to the skin and/or hair follicles is used. Thecomposition is applied three times per week over a five-year period.

It will be understood that modifications may be made without departingfrom the spirit of the present invention. Thus, the applicants'invention is understood to encompass not only the entire MI protein, aswell as methods of using and producing the same, but also activesegments of MI protein. Also, derivatives and analogs of MI protein canbe prepared by those skilled in the art, such as by making conservativechanges in one or more of the amino acids in the MI sequence.Additionally, non-peptide mimetics could be modeled after activesegments of MI protein, and thus these mimetics do not depart from thespirit of the present invention. Accordingly, the scope of the presentinvention should be considered in terms of the following claims, and itis understood not to be limited to that shown and described in thespecification.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 6                                                  (2) INFORMATION FOR SEQ ID NO: 1:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21                                                                (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ThrGlnThrValXaaAsnPheThrAspGlyAlaLeuValGlnHi                                  151015                                                                        GlnGluXaaXaaGlyLys                                                            20                                                                            (2) INFORMATION FOR SEQ ID NO: 2:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       LeuValValGluCysValMetAsnAsnValThrCysThrArgXa                                  151015                                                                        TyrGluLys                                                                     (2) INFORMATION FOR SEQ ID NO: 3:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 408 bases                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Unknown                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       ATGGCCACAGTTCAGCAGCTGGAAGGAAGATGGCGCCTGGTG42                                  MetAlaThrValGlnGlnLeuGluGlyArgTrpArgLeuVal                                    1510                                                                          GACAGCAAAGGCTTTGATGAATACATGAAGGAGCTAGGAGTG84                                  AspSerLysGlyPheAspGluTyrMetLysGluLeuGlyVal                                    152025                                                                        GGAATAGCTTTGCGAAAAATGGGCGCAATGGCCAAGCCAGAT12                                  GlyIleAlaLeuArgLysMetGlyAlaMetAlaLysProAsp                                    303540                                                                        TGTATCATCACTTGTGATGGTAAAAACCTCACCATAAAAACT16                                  CysIleIleThrCysAspGlyLysAsnLeuThrIleLysThr                                    455055                                                                        GAGAGCACTTTGAAAACAACACAGTTTTCTTGTACCCTGGGA21                                  GluSerThrLeuLysThrThrGlnPheSerCysThrLeuGLY                                    606570                                                                        GAGAAGTTTGAAGAAACCACAGCTGATGGCAGAAAAACTCAG25                                  GluLysPheGluGluThrThrAlaAspGlyArgLysThrGln                                    7580                                                                          ACTGTCTGCAACTTTACAGATGGTGCATTGGTTCAGCATCAG29                                  ThrValCysAsnPheThrAspGlyAlaLeuValGlnHisGln                                    859095                                                                        GAGTGGGATGGGAAGGAAAGCACAATAACAAGAAAATTGAAA33                                  GluTrpAspGlyLysGluSerThrIleThrArgLysLeuLys                                    100105110                                                                     GATGGGAAATTAGTGGTGGAGTGTGTCATGAACAATGTCACC37                                  AspGlyLysLeuValValGluCysValMetAsnAsnValThr                                    115120125                                                                     TGTACTCGGATCTATGAAAAAGTAGAATAA40                                              CysThrArgIleTyrGluLysValGlu                                                   130135                                                                        (2) INFORMATION FOR SEQ ID NO: 4:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 135                                                               (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       MetAlaThrValGlnGlnLeuGluGlyArgTrpArgLeuVal                                    1510                                                                          AspSerLysGlyPheAspGluTyrMetLysGluLeuGlyVal                                    152025                                                                        GlyIleAlaLeuArgLysMetGlyAlaMetAlaLysProAsp                                    303540                                                                        CysIleIleThrCysAspGlyLysAsnLeuThrIleLysThr                                    455055                                                                        GluSerThrLeuLysThrThrGlnPheSerCysThrLeuGLY                                    606570                                                                        GluLysPheGluGluThrThrAlaAspGlyArgLysThrGln                                    7580                                                                          ThrValCysAsnPheThrAspGlyAlaLeuValGlnHisGln                                    859095                                                                        GluTrpAspGlyLysGluSerThrIleThrArgLysLeuLys                                    100105110                                                                     AspGlyLysLeuValValGluCysValMetAsnAsnValThr                                    115120125                                                                     CysThrArgIleTyrGluLysValGlu                                                   130135                                                                        (2) INFORMATION FOR SEQ ID NO: 5:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Unknown                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       CAGCCCGCCCGCACC15                                                             GlnProAlaArgThr                                                               (2) INFORMATION FOR SEQ ID NO: 6:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Unknown                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       AAAAAAGAAAGAAACAGTATG21                                                       LysLysGluArgAsnSerMet                                                         15                                                                            __________________________________________________________________________

What is claimed is:
 1. A method of producing a protein for inhibitingmelanogenesis in pigmentary cells comprising the steps of:(a) graftingmammalian skin onto a live host; (b) permitting said mammalian skin toremain on said live host for a predetermined period of time; (c)removing said mammalian skin from said host; and (d) extracting saidprotein from said skin wherein said protein has a molecular weight inthe range of about 13 to about 14 kDa.
 2. The method of claim 1, whereinsaid host is a nude mouse.
 3. The method of claim 2, wherein themammalian skin is human skin.
 4. The method of claim 3, wherein saidhuman skin is permitted to remain on said host for at least two weeks.5. The method of claim 4, wherein said extracting of said proteincomprises the following steps:(a) extracting a skin protein extract fromsaid skin using physiological saline; fractionating said skin proteinextract by ion exchange chromatography (b) into at least two solutions,wherein at least one of said solutions comprises said protein; (c)subjecting one of said solutions comprising said protein totwo-dimensional SDS-PAGE to isolate said protein into a spot on apolyacrylamide gel; and (d) electroeluting said protein from said spot.6. The method of claim 5, wherein said protein has the following aminoacid sequence: SEQ ID NO:
 4. 7. A substantially pure protein forinhibiting melanogenesis in pigmentary cells, said protein having thefollowing amino acid sequence: SEQ ID NO: 4, or an active melanogenesisinhibiting segment, derivative or analog thereof.
 8. A substantiallypure protein for inhibiting melanogenesis in pigmentary cells, saidprotein having the following amino acid sequence: SEQ ID NO:
 4. 9. Amethod of controlling melanogenesis in pigmentary cells comprising thesteps of:(a) providing an effective amount of a melanogenic inhibitorcomprising a melanogenic inhibitor protein, or an active melanogenesisinhibiting segment, derivative or analog thereof, said protein havingthe following amino acid sequence: SEQ ID NO: 4; (b) combining saidmelanogenic inhibitor with a suitable carrier; and (c) applying saidcombined melanogenic inhibitor to pigmentary cells to be controlled. 10.The method of claim 9, wherein said applying step is performed byinjection.
 11. The method of claim 9, wherein said applying step isperformed by topical application.
 12. The method of claim 11, whereinsaid pigmentary cells are skin pigmentary cells.
 13. The method of claim11, wherein said pigmentary cells are hair pigmentary cells.
 14. Themethod of claim 10, wherein said melonogenic inhibitor is saidmelanogenic inhibitor protein.
 15. The method of claim 11, wherein saidmelonogenic inhibitor is said melanogenic inhibitor protein.
 16. Amethod of eradicating melanoma cells comprising the steps of:(a)providing an effective amount of a melanogenic inhibitor comprising amelanogenic inhibitor protein, or an active melanogenesis inhibitingsegment, derivative or analog thereof, said protein having the followingamino acid sequence: SEQ ID NO: 4; (b) combining said melanogenicinhibitor with a suitable carrier; and (c) applying said combinedmelanogenic inhibitor to melanoma cells to be eradicated.
 17. The methodof claim 16, wherein said melanogenic inhibitor is said melanogenicinhibitor protein.
 18. The method of claim 17, wherein said applyingstep is performed by injection.
 19. The method of claim 17, wherein saidapplying step is performed by topical application.